Hydrophobic Phe Research Articles

Design, synthesis and anticancer evaluation of novel arylhydrazones of active methylene compounds

Nerve growth factor (NGF) and its receptor, tropomyosin kinase receptor kinase type A (TrkA) is emerging as an important target for Glioblastoma (GBM) treatment. TrkA is the cancer biomarker majorly involved in tumor invasion and migration into nearby normal tissue. However, currently, available Trk inhibitors exhibit many adverse effects in cancer patients, thus demanding a novel class of ligands to regulate Trk signaling. Here, we exploited the role of TrkA (NTRK1) expression from the 651 datasets of brain tumors. RNA sequence analysis identified overexpression of NTRK1 in GBM, recurrent GBM as well in Oligoastrocytoma patients. Also, TrkA expression tends to increase over the higher grades of GBM. TrkA protein targeting hydrazone derivatives, R48, R142, and R234, were designed and their mode of interaction was studied using molecular docking and dynamic simulation studies. Ligands' stability and binding assessment reveals R48, 2 2-(2-(2-hydroxy-4-nitrophenyl) hydrazineylidene)-1-phenylbutane-1,3-dione, as a potent ligand that interacts well with TrkA's hydrophobic residues, Ile, Phe, Leu, Ala, and Val. R48- TrkA exhibits stable binding potentials with an average RMSD value <0.8 nm. R48 obeyed Lipinski's rule of five and possessed the best oral bioavailability, suggesting R48 as a potential compound with drug-likeness properties. In-vitro analysis also revealed that R48 exhibited a higher cytotoxicity effect for U87 GBM cells than TMZ with the IC50 value of 68.99 μM. It showed the lowest percentage of cytotoxicity to the non-cancerous TrkA expressing MEF cells. However, further SiRNA analysis validates the non-specific binding of R48, necessitating structural alteration for the development of R48-based TrkA inhibitor for GBM therapeutics.

High-temperature behavior of hyperthermostable Thermotoga maritima xylanase XYN10B after designed and evolved mutations.

A hyperthermostable xylanase XYN10B from Thermotoga maritima (PDB code 1VBR, GenBank accession number KR078269) was subjected to site-directed and error-prone PCR mutagenesis. From the selected five mutants, the two site-directed mutants (F806H and F806V) showed a 3.3-3.5-fold improved enzyme half-life at 100 °C. The mutant XYNA generated by error-prone PCR showed slightly improved stability at 100 °C and a lower Km. In XYNB and XYNC, the additional mutations over XYNA decreased the thermostability and temperature optimum, while elevating the Km. In XYNC, two large side-chains were introduced into the protein's interior. Micro-differential scanning calorimetry (DSC) showed that the melting temperature (Tm) dropped in XYNB and XYNC from 104.9 °C to 93.7 °C and 78.6 °C, respectively. The detrimental mutations showed that extremely thermostable enzymes can tolerate quite radical mutations in the protein's interior and still retain high thermostability. The analysis of mutations (F806H and F806V) in a hydrophobic area lining the substrate-binding region indicated that active site hydrophobicity is important for high activity at extreme temperatures. Although polar His at 806 provided higher stability, the hydrophobic Phe at 806 provided higher activity than His. This study generates an understanding of how extreme thermostability and high activity are formed in GH10 xylanases. KEY POINTS: • Characterization and molecular dynamics simulations of TmXYN10B and its mutants • Explanation of structural stability of GH10 xylanase.

Confocal Raman Spectroscopic Imaging for Evaluation of Distribution of Nano-Formulated Hydrophobic Active Cosmetic Ingredients in Hydrophilic Films.

Film-forming systems are highly relevant to the topical administration of active ingredients (AI) to the body. Enhanced contact with the skin can increase the efficacy of delivery and penetration during prolonged exposure. However, after the evaporation of volatile solvents to form a thin film, the distribution of the ingredient should remain homogenous in order to ensure the effectiveness of the formula. This is especially critical for the use of hydrophobic molecules that have poor solubility in hydrophilic films. In order to address this concern, hydroxyphenethyl esters (PHE) of Punica granatum seed oil were prepared as a nanosuspension stabilised by poloxamers (NanoPHE). NanoPHE was then added to a formulation containing polyvinyl alcohol (PVA) as a film forming agent, Glycerol as a plasticiser and an antimicrobial agent, SepicideTM HB. Despite their reliability, reference methods such as high-performance liquid chromatography are increasingly challenged due to the need for consumables and solvents, which is contrary to current concerns about green industry in the cosmetics field. Moreover, such methods fail to provide spatially resolved chemical information. In order to investigate the distribution of ingredients in the dried film, Confocal Raman imaging (CRI) coupled to Non-negatively Constrained Least Squares (NCLS) analysis was used. The reconstructed heat maps from a range of films containing systematically varying PHE concentrations highlighted the changes in spectral contribution from each of the ingredients. First, using NCLS scores it was demonstrated that the distributions of PVA, Glycerol, SepicideTM HB and PHE were homogenous, with respective relative standard deviations (RSD) of 3.33%, 2.48%, 2.72% and 6.27%. Second, the respective relationships between ingredient concentrations in the films and their Raman responses, and the spectral abundance were established. Finally, a model for absolute quantification for PHE was be constructed using the percentage of spectral abundance. The prepared %w/w concentrations regressed against predicted %w/w concentrations, displaying high correlation (R2 = 0.995), while the Root Mean Squared Error (0.0869% w/w PHE) confirmed the precision of the analysis. The mean percent relative error of 3.75% indicates the accuracy to which the concentration in dried films could be determined, further supporting the suitability of CRI for analysis of composite solid film matrix. Ultimately, it was demonstrated that nanoformulation of hydrophobic PHE provides homogenous distribution in PVA based film-forming systems independent of the concentration of NanoPHE used in the formula.

Structural studies of complexes of kallikrein 4 with wild-type and mutated forms of the Kunitz-type inhibitor BbKI.

Structures of BbKI, a recombinant Kunitz-type serine protease inhibitor from Bauhinia bauhinioides, complexed with human kallikrein 4 (KLK4) were determined at medium-to-high resolution in four crystal forms (space groups P3121, P6522, P21 and P61). Although the fold of the protein was virtually identical in all of the crystals, some significant differences were observed in the conformation of Arg64 of BbKI, the residue that occupies the S1 pocket in KLK4. Whereas this residue exhibited two orientations in the highest resolution structure (P3121), making either a canonical trypsin-like interaction with Asp189 of KLK4 or an alternate interaction, only a single alternate orientation was observed in the other three structures. A neighboring disulfide, Cys191-Cys220, was partially or fully broken in all KLK4 structures. Four variants of BbKI in which Arg64 was replaced by Met, Phe, Ala and Asp were expressed and crystallized, and their structures were determined in complex with KLK4. Structures of the Phe and Met variants complexed with bovine trypsin and of the Phe variant complexed with α-chymotrypsin were also determined. Although the inhibitory potency of these variant forms of BbKI was lowered by up to four orders of magnitude, only small changes were seen in the vicinity of the mutated residues. Therefore, a totality of subtle differences in KLK4-BbKI interactions within the fully extended interface in the structures of these variants might be responsible for the observed effect. Screening of the BbKI variants against a panel of serine proteases revealed an altered pattern of inhibitory specificity, which was shifted towards that of chymotrypsin-like proteases for the hydrophobic Phe and Met P1 substitutions. This work reports the first structures of plant Kunitz inhibitors with S1-family serine proteases other than trypsin, as well as new insights into the specificity of inhibition of medically relevant kallikreins.

Effect of active-site aromatic residues Tyr or Phe on activity and stability of glucose 6-phosphate dehydrogenase from psychrophilic Arctic bacterium Sphingomonas sp.

Cold-adapted enzymes maintain correct conformation at their active sites despite their intrinsically flexible structures. The psychrophilic Arctic bacterium Sphingomonas sp. PAMC 26621 has two glucose 6-phosphate dehydrogenase (G6PD) isozymes, SpG6PD1 involved in the Entner-Doudoroff pathway and SpG6PD2 in the oxidative pentose phosphate pathway. Structural modeling of SpG6PD1 showed that the hydroxyl group of Tyr177 participates in substrate binding by forming a hydrogen bond with the phosphate group of glucose 6-phosphate, whereas in SpG6PD2, a Phe residue is present in the corresponding position of Tyr177. In this study, we investigated how subtle differences in aromatic residues in the substrate-binding pocket of SpG6PD1 affect enzymatic activity and stability. Mutations of Tyr177 to Ala, His, Phe, and Trp caused increases in the rigidity of the SpG6PD1 structure. Particularly, mutants Y177F and Y177W showed increased thermal stabilities compared to wild-type (WT) but 3- and 15-fold lower catalytic efficiencies, respectively. However, mutants Y177A and Y177H became heat-labile at moderate temperatures. These results indicate that an aromatic residue (Tyr or Phe) is necessary for the substrate-binding pocket of SpG6PD1; Tyr with its hydroxyl group is preferred for enzymatic activity, whereas the more hydrophobic Phe is preferred for thermal stability. Substitutions of bulky Trp for Tyr or Phe at this position resulted in substantial loss of activity. Our study suggests that delicate adjustment of aromatic residues can regulate the activity and stability of psychrophilic G6PD isozymes involved in different metabolic pathways.

Identification of common and distinct features of ligand-binding sites in kernel and outlier lipocalins

Lipocalins are potential targets for drug development, and therefore it is important to understand the common and distinct features of their ligand-binding sites. Here, the sequence and structural features of ligand-binding sites in kernel and outlier lipocalins were analyzed and compared. Ligand-binding sites of kernel lipocalins were sequentially more diverse than outlier lipocalins. The ligand-binding sites of the two subgroup lipocalins were relatively hydrophobic and exhibited positive or neutral net-charges, well-correlated with the hydrophobic ligands with neutral or acidic functional groups. There was a propensity of three hydrophobic residues, Phe, Leu and Tyr in the ligand-interacting residues of kernel lipocalins, whereas outlier lipocalins showed relatively lower propensity. Finally, six and ten crucial ligand-interacting positions were identified in the kernel and outlier lipocalins, respectively. These identified features are expected to be a theoretical reference for the engineering and exploration of kernel and outlier lipocalins.

Effect of Amino Acid Substitutions on Biological Activity of Antimicrobial Peptide: Design, Recombinant Production, and Biological Activity.

Recently, antimicrobial peptides have been introduced as potent antibiotics with a wide range of antimicrobial activities. They have also exhibited other biological activities, including anti-inflammatory, growth stimulating, and anti-cancer activities. In this study, an analog of Magainin II was designed and produced as a recombinant fusion protein. The designed sequence contained 24 amino acid residues (P24), in which Lys, His, Ser residues were substituted with Arg and also, hydrophobic Phe was replaced with Trp. Recombinant production of P24 in Escherichia coli (E. coli) BL21 using pTYB21, containing chitin binding domain and intein sequence at the N-terminus of the peptide gene, resulted in 1 μg mL-1 product from culture. Chitin column chromatography, followed by online peptide cleavage with thiol reducing agent was applied to purify the peptide. Antimicrobial activity was evaluated using five bacteria strains including Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumonia, E. coli, and Pseudomonas aeruginosa. Designed AMP exhibited promising antimicrobial activities with low minimum inhibitory concentration, in the range of 64-256 µg/mL. P24 showed potent antimicrobial activity preferably against Gram-positive bacteria, and more potent than pexiganan as a successful Magainin II analog for topical infections. In general, further modification can be applied to improve its therapeutic index.

Polymerizable aggregation-induced emission dye for preparation of cross-linkable fluorescent nanoprobes with ultra-low critical micelle concentrations

In recent years, aggregation-induced emission (AIE) dyes based fluorescent organic nanoparticles (FONs) have achieved significant progress in various biomedical applications. In this work, we developed a covalent strategy to prepare biocompatible AIE-active dyes based cross-linked copolymers (MPC-POSS-PhE) via controllable reversible addition fragmentation chain transfer (RAFT) polymerization using zwitterionic 2-methacryloyloxyethyl phosphorylcholine (MPC), polymerizable AIE dye (named as PhE) and 8-vinyl polyoctahedral silsesquioxanes (POSS) as monomers. Due to the existence of hydrophilic MPC and hydrophobic PhE, the resultant copolymers will self-assemble into core-shell nanoparticles in aqueous solution with ultra-low critical micelle concentration (CMC). This could effectively overcome the drawbacks of non-crosslinked micelles and show more attractive properties and better performance for biomedical applications. Furthermore, the characterization results and biological assays demonstrated that the final MPC-POSS-PhE FONs show stable aqueous stability, uniform size and morphology, high water dispersity, desirable optical properties and low cytotoxicity. These remarkable properties make the resultant AIE-active nanoprobes great potential for biomedical applications.

The HOOK region of voltage-gated Ca2+ channel β subunits senses and transmits PIP2 signals to the gate.

The β subunit of voltage-gated Ca2+ (CaV) channels plays an important role in regulating gating of the α1 pore-forming subunit and its regulation by phosphatidylinositol 4,5-bisphosphate (PIP2). Subcellular localization of the CaV β subunit is critical for this effect; N-terminal-dependent membrane targeting of the β subunit slows inactivation and decreases PIP2 sensitivity. Here, we provide evidence that the HOOK region of the β subunit plays an important role in the regulation of CaV biophysics. Based on amino acid composition, we broadly divide the HOOK region into three domains: S (polyserine), A (polyacidic), and B (polybasic). We show that a β subunit containing only its A domain in the HOOK region increases inactivation kinetics and channel inhibition by PIP2 depletion, whereas a β subunit with only a B domain decreases these responses. When both the A and B domains are deleted, or when the entire HOOK region is deleted, the responses are elevated. Using a peptide-to-liposome binding assay and confocal microscopy, we find that the B domain of the HOOK region directly interacts with anionic phospholipids via polybasic and two hydrophobic Phe residues. The β2c-short subunit, which lacks an A domain and contains fewer basic amino acids and no Phe residues in the B domain, neither associates with phospholipids nor affects channel gating dynamically. Together, our data suggest that the flexible HOOK region of the β subunit acts as an important regulator of CaV channel gating via dynamic electrostatic and hydrophobic interaction with the plasma membrane.

Role of a Structurally Equivalent Phenylalanine Residue in Catalysis and Thermal Stability of Formate Dehydrogenases from Different Sources.

Comparison of amino acid sequences of NAD+-dependent formate dehydrogenases (FDH, EC 1.2.1.2) from different sources and analysis of structures of holo-forms of FDH from bacterium Pseudomonas sp. 101 (PseFDH) and soya Glycine max (SoyFDH) as well as of structure of apo-form of FDH from yeast Candida boidinii (CboFDH) revealed the presence on the surface of protein globule of hydrophobic Phe residue in structurally equivalent position (SEP). The residue is placed in the coenzyme-binding domain and protects bound NAD+ from solvent. The effects of amino acid changes of the SEP on catalytic properties and thermal stability of PseFDH, SoyFDH, and CboFDH were compared. The strongest effect was observed for SoyFDH. All eight amino acid replacements resulted in increase in thermal stability, and in seven cases, increase in catalytic constant was achieved. Thermal stability of SoyFDH after mutations Phe290Asp and Phe290Glu increased 66- and 55-fold, respectively. KM values of the enzyme for substrate and coenzyme in different cases slightly increased or decreased. In case of one CboFDH, the mutein catalytic constant increased and thermal stability did not changed. In case of the second CboFDH mutant, results were the opposite. In one PseFDH mutant, amino acid change did not influence the catalytic constant, but in three others, the parameter was reduced. Two PseFDH mutants had higher and two mutants lower thermal stability in comparison with initial enzyme. Analysis of results of SEP mutagenesis in FDHs from bacterium, yeast, and plant shows that protein structure plays a key role for effect of the same amino acid changes in equivalent position in protein globule of formate dehydrogenases from different sources.

Ligand Binding Pocket Formed by Evolutionarily Conserved Residues in the Glucagon-like Peptide-1 (GLP-1) Receptor Core Domain

Glucagon-like peptide-1 (GLP-1) plays a pivotal role in glucose homeostasis through its receptor GLP1R. Due to its multiple beneficial effects, GLP-1 has gained great attention for treatment of type 2 diabetes and obesity. However, little is known about the molecular mechanism underlying the interaction of GLP-1 with the heptahelical core domain of GLP1R conferring high affinity ligand binding and ligand-induced receptor activation. Here, using chimeric and point-mutated GLP1R, we determined that the evolutionarily conserved amino acid residue Arg(380) flanked by hydrophobic Leu(379) and Phe(381) in extracellular loop 3 (ECL3) may have an interaction with Asp(9) and Gly(4) of the GLP-1 peptide. The molecular modeling study showed that Ile(196) at transmembrane helix 2, Met(233) at ECL1, and Asn(302) at ECL2 of GLP1R have contacts with His(1) and Thr(7) of GLP-1. This study may shed light on the mechanism underlying high affinity interaction between the ligand and the binding pocket that is formed by these conserved residues in the GLP1R core domain.

Self-Assembly of Amphiphilic Peptide (AF)6H5K15 Derivatives: Roles of Hydrophilic and Hydrophobic Residues

A molecular dynamics simulation study is reported to investigate the roles of hydrophilic and hydrophobic residues in the self-assembly of (AF)6H5K15 peptide derivatives. The peptide, as well as water and counterions, are represented by the MARTINI coarse-grained model. The assembly is observed to follow a three-step process: formation of small clusters, large clusters, and micelles. With increasing length of hydrophilic Lys residues in (AF)6H5Kn (n = 10, 15, 20, and 25), assembly capability is found to be reduced with the formation of smaller micelles or the presence of individual peptide chains. Upon replacing Ala by more hydrophobic Phe in AmFnH5K15 (m + n = 12), larger micelles are formed. With increasing length of hydrophobic Phe residues in FnH5K15 (n = 4, 8, 12, and 16), micelle size increases and the morphology shifts from spherical to fiber-like. The simulation study provides mechanistic insight into the crucial roles of hydrophilicity and hydrophobicity in the assembly of (AF)6H5K15 derivatives; it reveals that assembly capability is reduced by increasing hydrophilicity, whereas increasing hydrophobicity leads to morphology transition.

Conformational Stability of Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Dictates Its Substrate Selectivity

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) converts inositol 1,3,4,5,6-pentakisphosphate(IP5) to inositol hexakisphosphate (IP6). IPK1 shares structural similarity with protein kinases and is suspected to employ a similar mechanism of activation. Previous studies revealed roles for the 1- and 3-phosphates of IP5 in IPK1 activation and revealed that the N-lobe of IPK1 is unstable in the absence of inositol phosphate (IP). Here, we demonstrate the link between IPK1 substrate specificity and the stability of its N-lobe. Limited proteolysis of IPK1 revealed that N-lobe stability is dependent on the presence of the 1-phosphate of the substrate, whereas overall stability of IPK1 was increased in ternary complexes with nucleotide and IPs possessing 1- and 3-phosphates that engage the N-lobe of IPK1. Thus, the 1- and 3-phosphates possess dual roles in both IPK1 activation and IPK1 stability. To test whether kinase stability directly contributed to substrate selectivity of the kinase, we engineered IPK1 mutants with disulfide bonds that artificially stabilized the N-lobe in an IP-independent manner thereby mimicking its substrate-bound state in the absence of IP. IPK1 E82C/S142C exhibited a DTT-sensitive 5-fold increase in kcat for 3,4,5,6-inositol tetrakisphosphate (3,4,5,6-IP4) as compared with wild-type IPK1. The crystal structure of the IPK1 E82C/S142C mutant confirmed the presence of the disulfide bond and revealed a small shift in the N-lobe. Finally, we determined that IPK1 E82C/S142C is substantially more stable than wild-type IPK1 under nonreducing conditions, revealing that increased stability of IPK1 E82C/S142C correlates with changes in substrate specificity by allowing IPs lacking the stabilizing 1-phosphate to be used. Taken together, our results show that IPK1 substrate selection is linked to the ability of each potential substrate to stabilize IPK1.

Sequence length determinants for self‐assembly of amphipathic β‐sheet peptides

Amphipathic peptides composed of alternating hydrophobic and hydrophilic amino acids are a privileged class of peptide, which have a high propensity to self-assemble into β-sheet fibrils. The Ac-(FKFE)2-NH2 peptide has been extensively studied and forms putative β-sheet bilayer fibrils in which the hydrophobic Phe side chains are organized to a single face of each constituent sheet; upon bilayer formation, these hydrophobic benzyl groups are sequestered in the hydrophobic core of the resulting fibril. In order for the Phe side chains to be uniformly displayed on one face of Ac-(FKFE)2-NH2 β-sheets, an antiparallel packing orientation in which one amino acid residue is unpaired must be adopted. Based on molecular models, we hypothesized that truncated seven amino acid derivatives of Ac-(FKFE)2-NH2 in which either the N-terminal Phe residue (Ac-KFEFKFE-NH2) or the C-terminal Glu residue (Ac-FKFEFKF-NH2) is eliminated should readily self-assemble into β-sheet bilayers in which all hydrogen bond and hydrophobic/charge interactions are satisfied. We found, however, that these minute changes in peptide sequence have unanticipated and dramatic effects on the self-assembly of each peptide. Ac-FKFEFKF-NH2 self-assembled into fibrils with unique morphology relative to the parent peptide, whereas the Ac-KFEFKFE-NH2 peptide had a strongly reduced propensity to self-assemble, even failing to self-assemble altogether under some conditions. These findings provide significant insight into the effect of sequence length and strand registry as well as hydrophobicity and charge on the self-assembly of simple amphipathic peptides to illuminate the possibility of tuning self-assembly processes and the resulting structures with minute changes to peptide sequence.

Manipulation of the Conformation and Enzymatic Properties of T1 Lipase by Site-Directed Mutagenesis of the Protein Core

In silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues, Val, Met, Leu, Ile, Trp, and Phe into Gln 114 of T1 lipase. The in silico investigations accurately predicted the enzymatic characteristics of the mutants in the experimental studies and provided rationalization for some of the experimental observations. Substitution with Leu successfully improved the conformational stability and enzymatic characteristics of T1 lipase. However, replacement of Gln114 with Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability, diminished lipase activity and inferior enzymatic characteristics. These results suggested that the substitution of a larger residue in a densely packed area of the protein core can have considerable effects on the structure and function of an enzyme. This is especially true when the residue is next to the catalytic serine as demonstrated with the Phe and Trp mutation.

Substrate specificity screening of oat (Avena sativa) seeds aminopeptidase demonstrate unusually broad tolerance in S1 pocket

Aminopeptidases are proteolytic enzymes that remove one amino acid at a time from N-terminus of peptidic substrates. In plants, inhibitors of aminopeptidases can find potential applications in agriculture as herbicides. In this report we have used a library of fluorogenic derivatives of natural and unnatural amino acids for substrate specificity profiling of oat (Avena sativa) aminopeptidase. Interestingly, we have found that this enzyme recognizes effectively among the natural amino acids basic residues like Arg and Lys, hydrophobic Phe, Leu and Met, but also to some extent acidic residues Asp and Glu. In the case of unnatural amino acids hydrophobic residues (hPhe and hCha) and basic hArg were preferentially recognized.

Molecular Docking Studies of Type III Secretion System Effector Sopb Homolog in Vibrio vulnificus

Pathogenic bacteria use the needle shaped Type III secretion system to inject effector proteins into the host cell. The SopB effector protein of Salmonella mediates invasion by evading the host immune response. Being a phosphoinositide phosphatase, it synthesizes phospholipids at the host cell membrane, after targeting host cell ubiquitin. Ubiquitination of SopB are known to control the biological activity of SopB at the plasma membrane. The identified SopB effector protein of Vibrio vulnificus which is a human pathogen found in the marine environment was homologous to SopB of Salmonella and E.coli. Structural superposition with available structure of SopB of Salmonella yielded a DNA linking domain similar to Salmonella SopB. Ubiquitination sites for SopB homolog in V.vulnificus was predicted by bioinformatics tools which was further supported by molecular docking studies. The ubiquitin binding sites were proposed to be structurally similar to the conserved ubiquitin binding motif of human polymerase complexed with ubiquitin (2KTF). The ubiquitin binding sites having Leu residue at 226, Leu 235 and Leu 234 were conserved whereas the hydrophobic Phe was replaced by Tyr at 223 in the sopB homolog of V.vulnificus. The conserved Glu 228 of SopB protein was predicted to be involved in imparting a electronegative potential in the ligand binding site. The ubiquitin molecule docked with SopB of V.vulnificus had Leu8 for binding interaction and recognition which was found to be similar to the ubiquitin-human polymerase complex. Thus the host immune response was predicted to be targeted by SopB effector in V.vulnificus by altering the ubiquitin pathway.

A mutational analysis and molecular dynamics simulation of quinolone resistance proteins QnrA1 and QnrC from Proteus mirabilis

BackgroundThe first report on the transferable, plasmid-mediated quinolone-resistance determinant qnrA1 was in 1998. Since then, qnr alleles have been discovered worldwide in clinical strains of Gram-negative bacilli. Qnr proteins confer quinolone resistance, and belong to the pentapeptide repeat protein (PRP) family. Several PRP crystal structures have been solved, but little is known about the functional significance of their structural arrangement.ResultsWe conducted random and site-directed mutagenesis on qnrA1 and on qnrC, a newly identified quinolone-resistance gene from Proteus mirabilis. Many of the Qnr mutants lost their quinolone resistance function. The highly conserved hydrophobic Leu or Phe residues at the center of the pentapeptide repeats are known as i sites, and loss-of-function mutations included replacement of the i site hydrophobic residues with charged residues, replacing the i-2 site, N-terminal to the i residues, with bulky side-chain residues, introducing Pro into the β-helix coil, deletion of the N- and C-termini, and excision of a central coil. Molecular dynamics simulations and homology modeling demonstrated that QnrC overall adopts a stable β-helix fold and shares more similarities with MfpA than with other PRP structures. Based on homology modeling and molecular dynamics simulation, the dysfunctional point mutations introduced structural deformations into the quadrilateral β-helix structure of PRPs. Of the pentapeptides of QnrC, two-thirds adopted a type II β-turn, while the rest adopted type IV turns. A gap exists between coil 2 and coil 3 in the QnrC model structure, introducing a structural flexibility that is similar to that seen in MfpA.ConclusionThe hydrophobic core and the β-helix backbone conformation are important for maintaining the quinolone resistance property of Qnr proteins. QnrC may share structural similarity with MfpA.

Angiotensin-I-converting enzyme inhibitory activity and bitterness of enzymatically-produced hydrolysates of shrimp ( Pandalopsis dispar) processing byproducts investigated by Taguchi design

The importance of water-to-substrate ratio, protease type, percent enzyme and incubation time on hydrolysates produced from shrimp processing byproducts was investigated using Taguchi’s L 16 (4 5) experimental design. Protease type significantly ( p < 0.05) influenced soluble yield, degree of hydrolysis (DH), angiotensin-I-converting enzyme (ACE) inhibitory activity and bitterness of hydrolysates, while percent enzyme only affected the DH. Hydrolysates produced by Alcalase and Protamex possessed strong ACE inhibitory activity ( IC 50 = 100–200 μg/ml and 70 μg/ml, respectively), accompanied by high yield, high DH and strong bitterness. Furthermore, ACE inhibition was positively correlated ( r 2 = 0.87) with bitterness of the hydrolysates. Fractionation by size-exclusion chromatography revealed that the bitter substances, which also showed strong ACE inhibition, were <3 kDa in size and contained many hydrophobic residues, including Tyr, Phe, Leu, Ile, Val and Lys. Despite the bitterness, these hydrolysates may have potential health benefits, arising from their potent ACE inhibitory activity.

Structural requirements of penetratin absorption enhancement efficiency for insulin delivery

Penetratin, a 16-residue peptide, is used widely as a highly efficient delivery carrier for a wide range of poorly permeable therapeutic cargoes. The crucial structural features of penetratin remain unclear, as demonstrated by the difficulties encountered in designing new molecules. The efficiency in enhancing nasal insulin absorption was compared between l-penetratin and 20 of its analogues in rats. We also measured lactate dehydrogenase (LDH) leakage as an indicator of cytotoxicity and scored the histopathological irritation. Substitution of a cationic residue (Arg or Lys) with Leu or addition of tetra-arginine to the C- or N-terminus of penetratin caused considerable reduction in the enhancing efficiency properties of the modified analogues. Mutual exchanging of Arg and Lys in corresponding analogues produced nearly inactive analogues, although changing Arg to Lys in the same analogue produced similar penetratin activity. In addition, activity was impaired markedly upon modification of penetratin within amphiphilic (Trp) or hydrophobic (Ile and Phe) residues. Chain size-modified analogues lacked the ability to induce nasal insulin absorption. In contrast, rearrangement of the modified analogues by C,N-half-exchange and reverse analogues produced activity similar to that of the original penetratin. The enhancing activity was inhibited almost completely upon sequence arrangement of the resulting analogues. Surprisingly, a shuffle (Arg, Lys fix) 2 analogue increased insulin absorption significantly, reaching a relative bioavailability value 1.85-times that of original penetratin. This analogue caused negligible release of LDH in nasal lavage fluid and maintained the integrity of the nasal respiratory epithelium. In conclusion, modulation of amino acid sequences by fixing the cationic residue positions can augment penetratin-enhanced nasal absorption and may lead to improvements in nasal insulin absorption.